1. Field
The present disclosure relates to xylanases and methods for their expression and use. Specifically, the disclosure is related to a thermophilic xylanase (Xyl-1) and homologs thereof derived from Acidothermus cellulolyticus, and the use of these enzymes in hydrolyzing lignocellulose.
2. Related Art
Lignocellulose is plant biomass composed of cellulose, hemicellulose, and lignin. Lignocellulose serves as an abundant and inexpensive source of fermentable biomass. However, one barrier to the utilization of lignocellulose is the tight crosslinking of the cellulose and hemicellulose to the lignin. Breaking down lignocellulose (i.e. separating cellulose and hemicellulose from lignin) is energy intensive, and thus inefficient. Efficient utilization of lignocellulosic biomass will enhance the economic competitiveness of bioconversion processes which must compete with petrochemical processes. It has been shown that xylanase enzymes can be used to efficiently break down lignocellulose.
Xylanase enzymes are important in a wide variety of biotechnological and industrial applications. These include prebleaching of kraft pulp in the pulp and paper industry, recovery of cellulose fiber in textiles, enhancing digestibility of animal feed and silage, clarification of juices and beer, separation of cereal gluten and starch, assorted applications in the bakery industry, as well as the production of xylo-oligosaccharides for pharmacological applications and food additives (1, 2, 9, 14, and 21). Furthermore, recent interest in biofuels production from lignocellulosic plant biomass has brought xylanases into renewed prominence (5). Collins et al. (4) recently reviewed the physicochemical and functional characteristics of xylanases from six different families, their mechanism of action, and industrial applications.
Xylanase production is commonly obtained from Trichoderma reesei and Trichoderma harzianum strain E58, both from the Forintek Canada Corp. culture collection. Although both fungi are prolific producers of extracellular xylanases, fungal growth and enzyme production can only be carried out at mesophilic temperatures (e.g., 28° C.). Consequently the fermentation requires considerable cooling water during fungal growth and is easily subjected to bacterial contamination. The xylanase enzymes produced are also thermally unstable, losing over 90% of their activities within a half hour of incubation at 50° C. As a result, enzymatic hydrolysis of lignocellulose using these enzymes has to be carried out at a lower temperature of about 37-45° C. This in turn lowers the hydrolysis efficiencies, necessitates asceptic conditions during hydrolysis, as well as preventing prolonged enzyme use without replacement. However, higher efficiency of hydrolysis can be obtained by using thermophilic xylanases.
Recently, several thermophilic xylanases from fungal and bacterial microorganisms have been identified (FIG. 1). For example, U.S. Pat. No. 5,935,836 discloses a thermophilic xylanase isolated from Actinomadura flexuosa that has an optimal pH of 6.0-7.0 and a temperature range of 70-80° C. In addition, U.S. Pat. No. 5,395,765 discloses a xylanase derived from Rhodothermus, having activity over a pH range of 5-8 and thermostability at temperatures from 85-100° C. However, a xylanase with a more acidic pH range is desired for the utilization of hemicellulose biomass in fermentation.
The thermophilic cellulolytic bacterium Acidothermus cellulolyticus is described in Mohagheghi et al. (12), and the production of cellulase is described in Shiang et al. (19). However, neither reference describes a purified xylanase that may be useful at a low pH and high temperatures. U.S. Pat. No. 5,902,581 discloses a xylanase derived from Acidothermus cellulolyticus that is active at a pH range from 3.6-4.2 and that is thermostable at a range of 70-80° C. However, this A. cellulolyticus xylanase does not have optimal activity at temperatures above 80° C. or at a pH range from 4.5-6.0.